Publications by authors named "Nora Saraco"

19 Publications

  • Page 1 of 1

Endocannabinoid signaling impairs syncytialization: Using flow cytometry to evaluate forskolin-induced cell fusion.

Placenta 2021 Jan 26;103:152-155. Epub 2020 Oct 26.

Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Centro de Estudios Farmacológicos y Botánicos (UBA-CONICET-CEFYBO), Facultad de Medicina, Buenos Aires, Argentina.

Cytotrophoblast cells fuse to form the syncytiotrophoblast, the main structure responsible for the placenta's specialized functions. This complex process denominated syncytialization is fundamental for a correct pregnancy outcome. We observed that the endocannabinoid anandamide disrupts syncytialization employing traditional techniques and flow cytometry in BeWo cell line.
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http://dx.doi.org/10.1016/j.placenta.2020.10.028DOI Listing
January 2021

Estrogens in Human Male Gonadotropin Secretion and Testicular Physiology From Infancy to Late Puberty.

Front Endocrinol (Lausanne) 2020 25;11:72. Epub 2020 Feb 25.

Endocrinology Department, Hospital de Pediatría "Prof. Dr. Juan P. Garrahan", Buenos Aires, Argentina.

Several reports in humans as well as transgenic mouse models have shown that estrogens play an important role in male reproduction and fertility. Estrogen receptor alpha (ERα) and beta (ERβ) are expressed in different male tissues including the brain. The estradiol-binding protein GPER1 also mediates estrogen action in target tissues. In human testes a minimal ERα expression during prepuberty along with a marked pubertal up-regulation in germ cells has been reported. ERβ expression was detected mostly in spermatogonia, primary spermatocytes, and immature spermatids. In Sertoli cells ERβ expression increases with age. The aromatase enzyme (cP450arom), which converts androgens to estrogens, is widely expressed in human tissues (including gonads and hypothalamus), even during fetal life, suggesting that estrogens are also involved in human fetal physiology. Moreover, cP450arom is expressed in the early postnatal testicular Leydig cells and spermatogonia. Even though the aromatase complex is required for estrogen synthesis, its biological relevance is also related to the regulation of the balance between androgens and estrogens in different tissues. Knockout mouse models of aromatase (ArKO) and estrogen receptors (ERKOα, ERKOβ, and ERKOαβ) provide an important tool to study the effects of estrogens on the male reproductive physiology including the gonadal axis. High basal serum FSH levels were reported in adult aromatase-deficient men, suggesting that estrogens are involved in the negative regulatory gonadotropin feedback. However, normal serum gonadotropin levels were observed in an aromatase-deficient boy, suggesting a maturational pattern role of estrogen in the regulation of gonadotropin secretion. Nevertheless, the role of estrogens in primate testis development and function is controversial and poorly understood. This review addresses the role of estrogens in gonadotropin secretion and testicular physiology in male humans especially during childhood and puberty.
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http://dx.doi.org/10.3389/fendo.2020.00072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051936PMC
March 2021

VIP Promotes Recruitment of Tregs to the Uterine-Placental Interface During the Peri-Implantation Period to Sustain a Tolerogenic Microenvironment.

Front Immunol 2019 8;10:2907. Epub 2020 Jan 8.

CONICET, Laboratorio de Inmunofarmacología, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Universidad de Buenos Aires, Buenos Aires, Argentina.

Uterine receptivity and embryo implantation are two main processes that need a finely regulated balance between pro-inflammatory and tolerogenic mediators to allow a successful pregnancy. The neuroimmune peptide vasoactive intestinal peptide (VIP) is a key regulator, and it is involved in the induction of regulatory T cells (Tregs), which are crucial in both processes. Here, we analyzed the ability of endogenous and exogenous VIP to sustain a tolerogenic microenvironment during the peri-implantation period, particularly focusing on Treg recruitment. Wild-type (WT) and VIP-deficient mice [heterozygous (HT, +/-), knockout (KO, -/-)], and FOXP3-knock-in-GFP mice either pregnant or in estrus were used. During the day of estrus, we found significant histological differences between the uterus of WT mice vs. VIP-deficient mice, with the latter exhibiting undetectable levels of FOXP3 expression, decreased expression of interleukin (IL)-10, and vascular endothelial growth factor (VEGF)c, and increased gene expression of the Th17 proinflammatory transcription factor RORγt. To study the implantation window, we mated WT and VIP (+/-) females with WT males and observed altered FOXP3, VEGFc, IL-10, and transforming growth factor (TGF)β gene expression at the implantation sites at day 5.5 (d5.5), demonstrating a more inflammatory environment in VIP (+/-) vs. VIP (+/+) females. A similar molecular profile was observed at implantation sites of WT × WT mice treated with VIP antagonist at d3.5. We then examined the ability GFP-sorted CD4+ cells from FOXP3-GFP females to migrate toward conditioned media (CM) obtained from d5.5 implantation sites cultured in the absence/presence of VIP or VIP antagonist. VIP treatment increased CD4+FOXP3+ and decreased CD4+ total cell migration towards implantation sites, and VIP antagonist prevented these effects. Finally, we performed adoptive cell transfer of Tregs (sorted from FOXP3-GFP females) in VIP-deficient-mice, and we observed that FOXP3-GFP cells were mainly recruited into the uterus/implantation sites compared to all other tested tissues. In addition, after Treg transfer, we found an increase in IL-10 expression and VEGFc in HT females and allowed embryo implantation in KO females. In conclusion, VIP contributes to a local tolerogenic response necessary for successful pregnancy, preventing the development of a hostile uterine microenvironment for implantation by the selective recruitment of Tregs during the peri-implantation period.
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http://dx.doi.org/10.3389/fimmu.2019.02907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960177PMC
November 2020

Accelerated Pubertal Tempo in a 46,XY Aromatase-Deficient Patient.

Horm Res Paediatr 2018 31;90(4):275-282. Epub 2018 Aug 31.

Servicio de Endocrinología, Hospital de Pediatría Garrahan, Buenos Aires, Argentina.

Background: Aromatase deficiency is a rare autosomal recessive disorder. 46,XY-affected patients often remain undiagnosed until late puberty. Only 2 pediatric cases have been reported. Data on pubertal development in affected males are scarce.

Aim: To report the clinical phenotype and hormonal studies of an aromatase-deficient boy during the prepubertal and early pubertal period.

Results: The patient was the older brother of a 46,XX girl with aromatase deficiency. Molecular analysis revealed a previously reported homozygous mutation (Arg192Cys) in the CYP19A1 gene. Pubertal onset was at 9.8 years. At 11.3 years of age, signs of rapidly progressive puberty were seen. Laboratory tests revealed normal pubertal basal and GnRH-stimulated gonadotropin levels, normal Sertoli cell markers, and increased testosterone. The prepubertal lumbar spine bone mineral density (BMD) was normal but pubertal bone mineral accrual was incomplete, leading to osteopenia.

Conclusion: Estrogen restraint on gonadotropin secretion has been demonstrated in animal and human models. Interestingly, our patient presented with accelerated puberty and apparently normal pituitary gonadal function. These findings suggest that aromatase activity may be required to define pubertal progression in boys. Estrogen deficiency due to aromatase deficiency is responsible for insufficient bone mineral accrual during puberty.
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http://dx.doi.org/10.1159/000492128DOI Listing
April 2019

DNA methylation is not involved in specific down-regulation of HSD3B2, NR4A1 and RARB genes in androgen-secreting cells of human adrenal cortex.

Mol Cell Endocrinol 2017 02 23;441:46-54. Epub 2016 Sep 23.

Endocrine Service-CONICET, Hospital de Pediatria Garrahan, Buenos Aires, Argentina; National Research Council of Argentina (CONICET), Argentina.

We hypothesized that DNA methylation is involved in human adrenal functional zonation. mRNAs expression and methylation pattern of RARB, NR4A1 and HSD3B2 genes in human adrenal tissues (HAT) and in pediatric virilizing adrenocortical tumors (VAT) were analyzed. For analysis of the results samples were divided into 3 age groups according to FeZ involution, pre and post-adrenarche ages. In all HAT, similar RARB mRNA was found including microdissected zona reticularis (ZR) and zona fasciculata, but HSD3B2 and NR4A1 mRNAs were lower in ZR (p < 0.05). NR4A1 and RARB promoters remained unmethylated in HAT and VAT. No adrenal zone-specific differences in NR4A1 methylation were observed. In summary, RARB was not associated with ZR-specific downregulation of HSD3B2 in postnatal human adrenocotical zonation. DNA methylation would not be involved in NR4A1 adrenocortical cell-type specific downregulation. Lack of CpG islands in HSD3B2 suggested that HSD3B2 ZR-specific downregulation would not be directly mediated by DNA methylation.
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http://dx.doi.org/10.1016/j.mce.2016.09.024DOI Listing
February 2017

An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

Horm Res Paediatr 2015 5;84(4):275-82. Epub 2015 Aug 5.

Endocrinology Service, Hospital de Pediatrx00ED;a Garrahan, Buenos Aires, Argentina.

Background/aims: Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues.

Methods: Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied.

Results: A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues.

Conclusions: The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient.
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http://dx.doi.org/10.1159/000437142DOI Listing
September 2016

Five new cases of 46,XX aromatase deficiency: clinical follow-up from birth to puberty, a novel mutation, and a founder effect.

J Clin Endocrinol Metab 2015 Feb 21;100(2):E301-7. Epub 2014 Nov 21.

Endocrinology Service (R.M., N.P.G., M.Co., G.G., M.J., P.R., D.M.W., M.Ci., M.A.R., A.B., N.S.), Laboratory of Cellular Biology and Retrovirus (C.R.), Hospital de Pediatria Garrahan, C1245AAM Buenos Aires, Argentina; Endocrine Service (G.P.), Hospital Infantil Municipal de Córdoba, 5000 Córdoba, Argentina; Endocrine Service (J.G.F.), Hospital Regional de Concepcion, CPT4146GXD Tucuman, Argentina; and Endocrine Service (M.M.), Hospital de Niños de la Santísima Trinidad de Córdoba, 5000 Córdoba, Argentina.

Context: Aromatase is the key enzyme for estrogen biosynthesis and is encoded by the CYP19A1 gene. Since 1991, several molecular CYP19A1 gene alterations associated with aromatase deficiency have been described in both sexes.

Objective: The objective of the study was to detect CYP19A1 mutations in five aromatase-deficient 46,XX patients, to describe the clinical follow-up from birth to puberty and to perform haplotype analysis associated with the high-frequency c.628G>A splice mutation in Argentinean patients.

Design: The design of the study was the sequencing of the coding and flanking intronic regions of the CYP19A1 gene in all patients and parents. Haplotype analysis of patients carrying the c.628G>A mutation was also performed.

Patients: Clinical and biochemical findings in five new cases and one previously reported female aromatase-deficient patient (46,XX) are described. All patients presented with ambiguous genitalia at birth. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency as well as other steroidogenic defects were ruled out.

Results: Phenotypic variability among the affected patients was found during follow-up. Direct sequencing of the CYP19A1 gene from genomic DNA revealed one novel mutation (c.574C>T) in two patients. In silico analysis predicted the c.574C>T mutation to be probably damaging. Four of six nonrelated patients presented with the c.628G>A splice mutation. Haplotype analysis showed that the c.628G>A splice mutation is associated with the same haplotype in our population.

Conclusions: Increased knowledge on phenotypical variability found in female aromatase-deficient patients is useful to improve the detection rate in this disorder. In our population, a genetic founder defect has probably contributed to an increase in the incidence of the c.628G>A splice mutation.
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http://dx.doi.org/10.1210/jc.2014-2967DOI Listing
February 2015

Preserved fertility in a patient with a 46,XY disorder of sex development due to a new heterozygous mutation in the NR5A1/SF-1 gene: evidence of 46,XY and 46,XX gonadal dysgenesis phenotype variability in multiple members of an affected kindred.

Horm Res Paediatr 2012 14;78(2):119-26. Epub 2012 Aug 14.

Endocrinology Service, Hospital de Pediatría Garrahan, Buenos Aires, Argentina.

In humans, steroidogenic factor 1 (NR5A1/SF-1) mutations have been reported to cause gonadal dysgenesis, with or without adrenal failure, in both 46,XY and 46,XX individuals. We have previously reported extreme within-family variability in affected 46,XY patients. Even though low ovarian reserve with preserved fertility has been reported in females harboring NR5A1 gene mutations, fertility has only been observed in one reported case in affected 46,XY individuals. A kindred with multiple affected members presenting gonadal dysgenesis was studied. Four 46,XY individuals presented severe hypospadias at birth, one of them associated with micropenis and cryptorchidism. The other 3 developed spontaneous male puberty, and 1 has fathered 5 children. Four 46,XX patients presented premature ovarian failure (one of them was not available for the study) or high follicle-stimulating hormone levels. Mutational analysis of the NR5A1 gene revealed a novel heterozygous mutation, c.938G→A, predicted to cause a p.Arg313Hys amino acid change. A highly conserved amino acid of the ligand-binding domain of the mature protein is affected, predicting abnormal protein function. We confirm that preserved fertility can be observed in patients with a 46,XY disorder of sex development due to heterozygous mutations in the NR5A1 gene.
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http://dx.doi.org/10.1159/000338346DOI Listing
February 2013

Three new SF-1 (NR5A1) gene mutations in two unrelated families with multiple affected members: within-family variability in 46,XY subjects and low ovarian reserve in fertile 46,XX subjects.

Horm Res Paediatr 2011 22;75(1):70-7. Epub 2010 Sep 22.

Endocrinology Service, Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

Background: Three novel heterozygous SF-1 gene mutations affecting multiple members of two unrelated families with a history of 46,XY disorders of sex development (DSD) and 46,XX ovarian insufficiency are described.

Methods: clinical and mutational analysis of the SF-1 gene in 9 subjects of two families.

Results: family 1 had 2 affected 46,XY DSD subjects. One, born with severe perineal hypospadias, was raised as a male, and presented normal adolescence. The other, born with ambiguous genitalia, uterus, and mild testicular dysgenesis, was raised as a female. A W279X heterozygous mutation and an intronic deletion (g3314-3317delTCTC (IVS 4 + 8) was found in the SF-1 gene. In family 2, 4/6 affected siblings had 46,XY DSD or hypospadias. An affected 46,XX sister had normal sexual development but increased FSH levels. The 37-year-old affected mother had entered menopause. An Y183X heterozygous mutation was detected.

Conclusion: an extreme within-family phenotypic variability, ranging from severe prenatal undervirilization to normal pubertal development, was observed in 46,XY-affected siblings, indicating that other unknown factors might be involved in the phenotype. Low ovarian reserve and preserved fertility in 46,XX subjects can be observed in heterozygous SF-1 gene mutations.
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http://dx.doi.org/10.1159/000320029DOI Listing
May 2011

Metformin, estrogen replacement therapy and gonadotropin inhibition fail to improve insulin sensitivity in a girl with aromatase deficiency.

Horm Res 2009 21;72(6):370-6. Epub 2009 Oct 21.

Endocrine Service, Hospital de Pediatría Garrahan, Buenos Aires, Argentina.

Background: Insulin resistance (IR), abnormal lipid profile, and other features of the metabolic syndrome have been described in CYP19 gene knockout mice and in aromatase-deficient adult men but not in prepubertal affected girls.

Aims: To study insulin sensitivity, as well as the effects of estrogen, metformin and GnRHa treatment on glucose homeostasis, in an aromatase-deficient girl.

Methods: Clinical, metabolic and hormonal follow-up data, from 8 to 12 years of age, is presented.

Results: At 9 years of age, IR (HOMA 5.6) and glucose intolerance was detected, along with high serum testosterone (2.28 nmol/l), androstenedione (4.92 nmol/l) and FSH (13.4 mIU/ml) levels. Estrogen replacement was ineffective to suppress gonadotropin and androgen levels, as well as IR. Under metformin therapy, she developed type 2 diabetes and acanthosis nigricans. GnRHa administration for 1 year resulted in marked decreases in gonadotropin and serum androgens, but severe IR persisted.

Conclusion: Postnatal estrogen replacement and a marked decrease of endogenous androgens failed to improve IR and glucose tolerance. We propose that, in females, the increment of androgens and/or lack of estrogens during fetal life might alter the mechanism of fetal programming of insulin sensitivity.
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http://dx.doi.org/10.1159/000249165DOI Listing
February 2010

Role of IGFs and insulin in the human testis during postnatal activation: differentiation of steroidogenic cells.

Pediatr Res 2008 Jun;63(6):662-6

Endocrine Service, Hospital de Pediatria Garrahan, Buenos Aires C1245 AAM, Argentina.

Immunoexpression of IGF-I, IGF-II, type 1 IGF receptor (IGFR), insulin receptor (IR), and GH receptor (GHR) was analyzed in human testis, in three age groups (Gr): Gr1 (neonates), Gr2 (postnatal testicular activation), and Gr3 (early prepuberty). In interstitial cells, low IGF-I and GHR, but moderate IR immunoexpression was observed in all Grs. However, high expression of IGF-II in Gr1, and moderate expression of IGFR in Gr1 and Gr2 were found. In Leydig cell (LC), high expression of IGF-II, moderate expression of IGFR and GHR, and undetectable IGF-I was found. Moreover, IR was highly expressed in Gr2. The effect of IGF-I on cell proliferation (PI) and apoptosis (AI), induction of cytochrome P450 side chain cleavage (cP450scc) immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA and testosterone (T) secretion was evaluated in human testis cell cultures. IGF-I increased P450scc immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA, T secretion, and PI, but decreased AI. We propose that IGF-II, mainly through IR, is involved in functional LC differentiation. In some interstitial cells, probably in LC precursors, IGF-II/IR could be involved, among other factors, in the stimulation of PI and/or inhibition of AI, and in LC differentiation.
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http://dx.doi.org/10.1203/PDR.0b013e31816c8ffcDOI Listing
June 2008

[Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans].

Medicina (B Aires) 2007 ;67(4):369-73

Laboratorio de Investigación, Hospital Garrahan, Buenos Aires, Argentina.

P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.
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September 2008

The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues.

Clin Endocrinol (Oxf) 2007 Nov 2;67(5):698-705. Epub 2007 Jul 2.

Molecular Biology Laboratory, Endocrinology Department, J. P. Garrahan Paediatric Hospital, Buenos Aires, Argentina.

Objective: The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated.

Design: To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues.

Patients: An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed.

Results: The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues.

Conclusions: Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.
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http://dx.doi.org/10.1111/j.1365-2265.2007.02948.xDOI Listing
November 2007

Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues.

J Clin Endocrinol Metab 2007 Jun 3;92(6):2215-22. Epub 2007 Apr 3.

Endocrinology Service, Hospital de Pediatria Garrahan, C de los Pozos 1881, Buenos Aires, Argentina.

Context: The mechanisms of postnatal adrenal zonation remain unclear.

Objective: To provide a clue for a possible role of estrogens in adrenarche, we studied the expression of estrogen receptor (ER)alpha, ERbeta, G protein-coupled receptor (GPR)30, and cP450aromatase (cP450arom) in human adrenal tissue.

Design: Human adrenal tissue was collected from three postnatal age groups (Grs): Gr 1, younger than 3 months (n = 12), fetal zone involution; Gr 2, 3 months to 6 yr (n = 17), pre-adrenarche; and Gr 3, older than 6-20 yr (n = 12), post-adrenarche period.

Results: ERbeta mRNA in Grs 1 and 3 was higher than in Gr 2 (P < 0.05). By immunohistochemistry and laser capture microdissection followed by RT-PCR, ERbeta was expressed in zona reticularis and fetal zone, GPR30 in zona glomerulosa (ZG) and adrenal medulla, while ERalpha mRNA and protein were undetectable. cP450arom mRNA in Gr 3 was higher than in Grs 1 and 2 (P < 0.05), and localized to ZG and adrenal medulla by laser capture microdissection. cP450arom Immunoreactivity was observed in adrenal medulla in the three Grs and in subcapsular ZG of Gr 3. Double-immunofluorescence studies revealed that cP450arom and chromogranin A only colocalize in adrenal medulla of subjects younger than 18 months. In these samples, exon 1.b-derived transcript was 3.5-fold higher, while exon 1.a-, 1.c-, and 1.d-derived transcripts were 3.3-, 1.9-, and 1.7-fold lower, respectively, than in subjects older than 6 yr.

Conclusions: Our results suggest that estrogens produced locally in adrenal medulla would play a role in zona reticularis functional differentiation through ERbeta. The cP450arom and GPR30 expression in subcapsular ZG, colocalizing with a high-cell proliferation index, previously reported, suggests a local GPR30-dependent estrogen action in proliferation and migration of progenitor adrenal cells.
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http://dx.doi.org/10.1210/jc.2006-2329DOI Listing
June 2007

Expression of aromatase, estrogen receptor alpha and beta, androgen receptor, and cytochrome P-450scc in the human early prepubertal testis.

Pediatr Res 2006 Dec 25;60(6):740-4. Epub 2006 Oct 25.

Research Laboratory, Hospital de Pediatria Garrahan, Buenos Aires C124 5AAM, Argentina.

The expression of aromatase, estrogen receptor alpha (ERalpha) and beta (ERbeta), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERalpha by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERbeta and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERbeta, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERbeta were immunoexpressed strongly whereas no expression of ERalpha, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.
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http://dx.doi.org/10.1203/01.pdr.0000246072.04663.bbDOI Listing
December 2006

High TGFbeta1, estrogen receptor, and aromatase gene expression in a large cell calcifying sertoli cell tumor (LCCSCT): implications for the mechanism of oncogenesis.

Pediatr Dev Pathol 2006 May-Jun;9(3):181-9

Laboratorio de Investigación, Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

Large cell calcifying Sertoli cell tumors (LCCSCT) are associated with Carney complex and Peutz-Jeghers syndrome. The mechanisms linking these 2 genetic defects to the genesis of this tumor are obscure. Studies of CYP19 (aromatase) and transforming growth factor (TGF)-beta1 messenger RNA (mRNA) abundance, estrogen receptor (ER), TGFbeta1, and TGFbeta type II receptor (R) immunochemistry were carried out in the testis of a patient with this tumor to gain information on possible mechanisms of cell tumor development. Testicular tissue of a prepubertal patient, collected at gonadectomy, was separated into 2 macroscopically distinct fractions: tumoral nodules (Tu) and extratumoral, normal-looking testicular tissue (ExTu). The patient was a 9.5-year-old boy with a 5-year history of bilateral gynecomastia (Tanner stage 4), no pubic hair, incipient genital development, and bilateral testicular nodules. Multiple pigmented lesions of the skin were present. Bilateral mammectomy and gonadectomy was performed. RNA was extracted from Tu and ExTu for semiquantitative reverse transcriptase-polymerase chain reaction of CYP19 and TGFbeta1. Protein expression of ER, TGFbeta1, and TGFbeta type II R in Tu and ExTu was detected by immunohistochemistry. Cell proliferation was estimated by Ki-67 antigen immunochemistry and apoptosis using a modified TUNEL assay. Mean expression of aromatase and TGFbeta1 mRNAs in Tu was 6- and 2.3-fold higher than in ExTu, respectively (P<0.05). Tumoral cells exhibited ER staining with a predominant extranuclear localization. Positive staining of Sertoli cells in Tu was higher than in ExTu. TGFbeta1 immunostaining of the interstitial cells in Tu was higher than in ExTu. TGFbeta type II R immunostaining was detected in most Sertoli and interstitial cells, but intensity in ExTu was lower than in Tu. No significant difference was detected in the proliferation index, but in Tu, the percentage of Sertoli cells in apoptosis (1.4%) was significantly lower (P<0.01) than in ExTu (14.0%). The following hypothesis is proposed. The congenital gene defects of Carney complex or of Peutz-Jeghers syndrome might trigger a cascade of intracellular events that leads to overexpression of aromatase in Sertoli cells, favoring the development of a LCCSCT. At some point in the evolution of the disease, a mutational event might induce a higher expression of the ER. Also, TGFbeta1 protein expression is increased in neighboring cells. In this environment, TGFbeta1 might switch from tumor suppressor to oncogenic factor and, along with estrogen-ER complexes, might favor tumor progression by inhibiting apoptosis.
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http://dx.doi.org/10.2350/06-04-0074.1DOI Listing
October 2006

Expression of the IGF system in human adrenal tissues from early infancy to late puberty: implications for the development of adrenarche.

Pediatr Res 2005 Sep;58(3):451-8

Laboratorio de Investigacion, Hospital de Pediatria Garrahan, Buenos Aires Argentina.

IGF-1, IGF-2, and type 1 IGF receptor (IGF-R1) mRNA expression and immunolocalization and cell proliferation index were studied in human adrenals from early infancy to late puberty. Adrenals were obtained from transplantation donors or from necropsies of endocrinologically normal subjects. Subjects were divided into three age groups: group 1, <3 mo of age, involution of fetal adrenals; group 2, 3 mo to 6 y of age, preadrenarche; and group 3, older than 6 y up to 20 y of age, postadrenarche. Cell proliferation index (Ki-67) in the outer, subcapsular, zona glomerulosa was significantly higher than in zona fasciculata of all groups and in zona reticularis or fetal zone. IGF-1 mRNA (semiquantitative reverse transcriptase-PCR and Northern blot) in group 2 was significantly higher than in group 1 and group 3 (p < 0.05). IGF2 mRNA in group 1 was significantly higher than in the other groups. IGF-R1 mRNA in group 3 was significantly higher than in group 2 but not different from group 1. Strong IGF-1, IGF-2, and IGF-R1 immunostaining signal was observed in the outer, subcapsular, zona glomerulosa and in zona fasciculata in the three groups, whereas a very weak IGF-1 and IGF-R1 immunostaining signal was found in fetal zone cells of group 1 and in zona reticularis of group 3. We propose that IGF-1 could be a factor involved in the postnatal mechanism of progenitor adrenal cell proliferation and migration. Our data also suggest that IGF-1 is not a direct regulatory factor of adrenal androgen production by zona reticularis cells.
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http://dx.doi.org/10.1203/01.PDR.0000179392.59060.93DOI Listing
September 2005

Temporal pattern of the induction of SF-1 gene expression by the signal transduction pathway involving 3',5'-cyclic adenosine monophosphate.

Acta Biochim Pol 2005 15;52(2):485-91. Epub 2005 May 15.

Department of Biochemistry and Molecular Biology, University of Medical Sciences, Poznań, Poland.

The objective of our study was to investigate the effect of stimulation of the cAMP-dependent pathway on the expression of an orphan nuclear receptor, SF-1/Ad4BP in mouse adrenal tumour, Y-1 cells in culture. We evaluated the temporal pattern of the effects of corticotropin (ACTH) and the adenylyl cyclase activator forskolin on the level of SF-1 mRNA, and compared the time course of induction of SF-1 with that of CYP11A1. Forskolin, corticotropin and 8-Br-cAMP significantly elevated the level of the SF-1 transcript, after 1.5 h of incubation, with a concomitant increase of SF-1 protein level, observed after 6 h. The CYP11A1 transcript increased gradually over the incubation period, and reached the maximal level after 12 to 24 h. The steady-state level of the SF-1 transcript was unaffected by forskolin when the cells were incubated with actinomycin D, indicating that stimulation of the cAMP pathway results in enhanced transcription of the gene. The effect of forskolin was augmented by cycloheximide, suggesting that an inhibitory protein, whose synthesis was inhibited by cycloheximide, could be involved in negative regulation of SF-1 expression. It is concluded that SF-1 expression is positively regulated by the cAMP pathway at the transcriptional level, and can represent the primary event in cAMP-mediated induction of steroid hormone synthesis in Y-1 cells.
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August 2009

Hypothalamic-pituitary-ovarian axis during infancy, early and late prepuberty in an aromatase-deficient girl who is a compound heterocygote for two new point mutations of the CYP19 gene.

J Clin Endocrinol Metab 2003 Nov;88(11):5127-31

Laboratorio de Investigación, Hospital de Pediatría Garrahan, Buenos Aires, Argentina 1245.

A loss of function mutation of the CYP19 aromatase gene leads to excess circulating androgens in the fetus and in the mother, resulting in ambiguous genitalia in the female fetus. Later on, lack of aromatase is responsible for sexual infantilism, primary amenorrhea, tall stature, and multicystic ovaries, even in preadolescent girls. Up to now, 11 CYP19 aromatase point mutations and 10 well-documented cases have been reported. In the present case, we are reporting the clinical and hormonal follow-up, from birth to 7 yr of age, of an affected girl with ambiguous genitalia. Gene analysis showed that she was a compound heterozygote for two new CYP19 aromatase point mutations. In the father's allele, there was a consensus 5' splice donor sequence mutation, GAA-AAA at cDNA position bp 655 in exon 5, which probably results in a cryptic donor site. In the mother's allele, there was a base A deletion in exon 9 (Delta A GLU 412X), causing a frame shift mutation, and a stop codon after 98 bp (33 codons) downstream, altering the critical heme-binding region. Basal serum LH and FSH levels were high at 8 d of age (42.9 and 51.3 U/liter), 26 d of age (76.2 and 119 U/liter), and 60 d of age (58.7 and 150 U/liter, respectively). Both gonadotropins dropped dramatically between the second and fifth months of age (to 1.79 and 14.9 U/liter) but remained higher than in normal control girls (0.64 and 8.5 U/liter, respectively). Serum testosterone (T) and androstenedione (Delta(4)A) levels were high during the first month, but Delta(4)A was normal at 2 months of age. However, at 5 months of age, along with significant decrements of serum LH and FSH levels and increments in serum Delta(4)A and T levels, a large ovarian cyst was removed from each gonad. Relatively high levels of T [27.3 ng/ml (94.6 nmol/liter); control, 34.9 ng/ml (121 nmol/liter)], but not of estradiol [1.8 ng/ml (6.6 nmol/liter); control 62.9 ng/ml (231 nmol/liter)], and a high T/estradiol ratio [15.2; control < 1] were found in the follicular fluid. Serum Delta(4)A and T levels remained normal from 1-5 yr of age, but they were high at the last visit (late prepuberty). A GnRH test was performed at 3.9, 6, and 7.1 yr of age. At 3.9 yr, a low prepubertal serum LH peak (2.12 U/liter) was found, but at the older ages, higher serum LH peaks (8.25 and 22.5 U/liter, respectively) were observed. Growth pattern and body mass index were normal, but after the age of 5.2 yr, delays in bone age greater than 2 yr were observed. We concluded that: 1) these two new CYP19 aromatase gene mutations are responsible for the phenotype of aromatase deficiency; 2) in girls, aromatase deficiency results in a decrease of the negative feedback of both serum LH and FSH, which can be detected as early as the second week after birth and persists up to the sixth month of life, and of FSH during the rest of prepuberty; and 3) because large ovarian cysts developed when serum LH and FSH dropped, aromatization of androgens might be required to prevent formation of cystic ovaries.
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http://dx.doi.org/10.1210/jc.2003-030433DOI Listing
November 2003